Project Summary/Abstract The long-term goals of our laboratory are to understand biological functions of proteins encoded by polycystic kidney disease (PKD) genes such as PKD1/polycystin-1 (PC1) and to determine PKD pathogenic pathways when they are mutated. This proposal is build on our previous discovery of cis-autoproteolytic cleavage of PC1 at the GPS motif and its central role in regulating PC1's biogenesis, trafficking and function. Our central hypothesis is that the GPS motif and the adjacent linker form a bipartite force-transduction module that mediates key functions of PC1. The goal of this project is to use a combination of molecular, biochemical, cellular, and biophysical methods as well as mouse models to dissect the role of the GPS-linker module in PC1 trafficking and function. Our Aims are: 1) Test the hypothesis that a tight association of the ?1-strand within the GPS is required for PC1 ciliary trafficking and function, and is disrupted by PKD1 mutations. We predict that tight association of this ?-strand within the GPS motif is required to enable PC1 to traffic to cilia and to induce in vitro tubulogenesis in MDCK cells, and is disrupted by PKD1 mutations; 2) Test the hypothesis that high rigidity and short length in the linker is required for PC1 ciliary trafficking and function, and is disrupted by PKD1-associated mutations. We predict that the rigidity of the linker is required to enable PC1 to traffic to cilia and to induce in vitro tubulogenesis in MDCK cells, and is disrupted by PKD1 mutations; and 3) Determine the in vivo role of the GPS-Linker module for trafficking and function of PC1 in the mouse kidney during embryonic and postnatal development. We will generate two new Pkd1 knockin mouse models affecting the GPS or the linker respectively. We will compare their phenotypes and assess the expression, cleavage, and trafficking of the mutant PC1 proteins in various developmental stages and nephron segments. These analyses should provide mechanistic insights into how the two components of the module operate together to regulate both forms of PC1 during embryonic and postnatal stages of development, and how their dysfunction might lead to PKD. Overall, we anticipate that the proposed studies should lead to a better understanding of the fundamental mechanisms that regulate PC1 function in kidney health and PKD, and might provide rationales for novel strategies to target the disease by manipulating the force-transduction process in polycystin-1.